This invention relates to a method for the quantitative determination or assay of a substance present in a biological fluid, especially in human blood serum. More particularly, the invention relates to a method in which a compound produced in the course of the determination is oxidized in an enzymatic oxidation-reduction reaction with simultaneous production of reduced beta-nicotinamide adenine dinucleotide in an amount proportional to the content of the substance in the fluid, and to an improvement for obviating the interference caused by another substance in the fluid which while not related quantitatively to the substance being determined undergoes an enzymatic oxidation-reduction reaction with the production of reduced beta-nicotinamide adenine dinucleotide and thus reduces the accuracy of the determination. The invention also relates to a reagent combination for use in the method.
A number of metabolites present in biological fluids may be determined advantageously by methods in which beta-nicotinamide adenine dinucleotide in its oxidized form (NAD), is converted in an enzymatic oxidation-reduction reaction to its reduced form (NADH) proportionally to the content of the substance being determined. The NADH may be determined quantitatively by measurement of the intensity of light absorption, preferably at the absorption maximum of 340 nanometers (nm.). Alternatively, the NADH produced may be reacted with 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride (INT) in an enzymatic oxidation-reduction reaction, to convert the INT to its reduced form (INTH). The INTH may be determined quantitatively by measurement of light absorption, preferably at the absorption maximum of 500 nm.
Substances which may be determined by the foregoing procedure include the enzyme alpha-amylase, transaminase enzymes including glutamate oxalacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT), and triglycerides. The procedure is especially useful for making determinations in human blood serum, including the serum content of plasma, and also in other biological fluids, including, for example, urine, duodenal fluid, and peritoneal fluid.
The foregoing procedure potentially is very attractive. However, the biological fluids contain endogenous materials other than the substances being determined, which themselves or the derivatives of which as produced during the determination participate in enzymatic oxidation-reduction reactions with the formation of NADH, thereby interfering with the determinations. As an example, alpha-amylase in a biological fluid, particularly human blood serum, is determined in existing procedures by allowing the enzyme to hydrolyze starch to maltose, after which the maltose is enzymatically hydrolyzed to glucose, the glucose is converted to glucose-6-phosphate, and the latter compound is reacted with NAD in an oxidation-reduction reaction to produce NADH. Endogenous glucose also present in the fluid participates in the latter reactions with the production of non-specific NADH, not related to the alpha-amylase activity, which reduces the accuracy of the determination to the extent of its presence.
While methods have been proposed for removing the endogenous glucose, as described in Clin. Chem., 21, 694 (1975) and Clin. Chem., 21, 947 (1975), they suffer from disadvantages, including, inter alia, lack of simiplicity and convenience as desired for laboratory procedures, and, particularly, for use as an emergency hospital diagnostic tool.
U.S. Pat. No. 4,000,042 discloses a method in which endogenous glucose initially is consumed, with the production of NADH, after which alpha-glucosidase is added to initiate the assay reactions. Employing such a method, it is possible for the absorbance due to alpha-amylase activity to be negligible compared to the absorbance due to endogenous glucose, thus leading to inaccurate results. Also, many of the available instruments for measuring absorbances are incapable of measuring high absorbances such as can be obtained with high endogenous glucose concentrations.
As another example, the activity of GOT and GPT in serum may be determined, based on the measurement of the concentration of glutamate formed in transaminase reactions, by oxidative deamination with simultaneous reduction of NAD to NADH, followed by reduction of INT to INTH. The reactions are performed by incubation of the serum with a single mixed reagent. However, endogenous amino acids in the serum likewise are oxidatively deaminated with the formation of non-specific NADH. While procedures are available to correct for such unreliable results, they involve disadvantages such as increased labor, lack of adaptability to performance in automated instruments, and/or incomplete removal of interfering endogenous materials.
An additional example is the determination of triglycerides (fatty acid esters of glycerol) in serum, wherein the triglycerides are enzymatically hydrolyzed with lipase to produce glycerol, and the glycerol or glycerol phosphate produced therefrom is reacted with NAD in an oxidation-reduction reaction to produce NADH. The results are adversely affected by the presence of endogenous glycerol, which is present in varying amounts in serum. While the endogenous glycerol content can be determined using a reagent containing all of the reactants except the lipase, and subtracting the value thus obtained from the value obtained using a complete reagent including lipase, the procedure is complicated and time-consuming.